Identification and antagonistic mechanism of biocontrol bacteria against Rhizoctonia solani
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摘要: 【目的】筛选对烟草靶斑病菌具有生防潜力的拮抗细菌，为烟草靶斑病的生物防治提供菌种资源。【方法】以立枯丝核菌为目标病原菌，采用稀释涂布平板法从广西健康烤烟根际土壤中分离、纯化细菌；采用平板对峙法筛选拮抗效果较好的菌株，测定抑菌效果；扫描电镜和超景深三维显微镜观察拮抗菌株处理立枯丝核菌菌丝的形态变化及菌落形态，并测定生理生化特征；采用细菌通用引物16S rDNA序列及gyrA基因对拮抗菌株DNA进行PCR扩增，采用NCBI的BLAST对测序结果同源对比分析，并基于亲缘关系较近的序列及模式菌株构建系统发育进化树；采用双平板对扣法初步探索拮抗菌株挥发性物质抑菌作用；利用顶空固相微萃取和气相色谱—质谱对拮抗菌株挥发性物质进行鉴定；通过对K326烤烟品种盆栽试验鉴定拮抗菌株对烟草靶斑病的防治效果。【结果】筛选到1株具有生防作用的拮抗菌株GXLL3319，结合该菌株菌落形态和生理生化特征及系统发育进化分析结果将该菌株鉴定为枯草芽孢杆菌（Bacillus subtilis）。该菌株对立枯丝核菌、烟草炭疽菌和尖孢镰刀菌3种烟草病原真菌抑制率均在50%以上；该菌株及其挥发性物质对立枯丝核菌抑制率分别为69.70%和59.22%。扫描电镜结果显示，经GXLL3319处理后的立枯丝核菌菌丝生长受到明显抑制，菌丝出现断裂、膨大和消解等现象。挥发性物质鉴定出31种抑菌活性物质，包括酮类、醇类、醛类、杂环类、硫化物、有机酸类、酯类、烷烃类和酚类。盆栽试验结果表明，GXLL3319菌液能有效抑制烟草靶斑病的发生，相对防效达66.07%，与15%井冈霉素A可溶粉剂的相对防效差异不显著（P>0.05）。【结论】枯草芽孢杆菌GXLL3319能有效抑制立枯丝核菌菌丝生长，其产生的挥发性物质包含多种抑菌活性物质，GXLL3319对烟草靶斑病有较好的防治效果，具有开发成为烟草靶斑病生防菌的潜力。Abstract: 【Objective】To screen antagonistic bacteria with biocontrol potential against Rhizoctonia solani,to provide strains for biocontrol on tobacco target spot.【Method】The dilution coated plate method was used to isolate and purify the bacteria from the rhizosphere soil of healthy flue-cured tobacco in Guangxi,with R. solani as the target pathogen. The plate confrontation method was used to screen the strains with good antagonistic effect,and the antibacterial effect was determined. Scanning electron microscopy and ultra depth of field 3D microscope was used to observe the morphological changes of mycelia of R. solani that treated with antagonistic strains and colony morphology,and measured its physiological and biochemical characteristics. PCR amplification was performed on the DNA of antagonistic strains using universal bacterial primers 16S rDNA sequences and gyrA gene. The sequencing results were compared and analyzed for homology using BLAST from NCBI. A phylogenetic tree was constructed based on sequences and type strains with close genetic relationships. The antibacterial effect of volatile organic compounds(VOCs)of antagonistic strains was preliminarily explored by double-plate buckle method. The volatile compounds of antagonistic strains were identified by headspace solidphase microextraction(HS-SPME)and gas chromatography-mass spectrometry(GC-MS). Identified the control effect of antagonistic strains on tobacco target spot disease through pot experiments on K326 tobacco variety.【Result】An antagonistic strain GXLL3319 with biocontrol activity was screened,and combined with the colony morphology,physiological and biochemical characteristics,and phylogenetic analysis results,the strain was identified as Bacillus subtilis. The inhibition rate of this strain on three kinds of tobacco pathogenic fungi,namely R. solani,Colletotrichum nicotianae and Fusarium oxysporum,was above 50%. The inhibition rate of this strain and its volatile substances on R. solani was 69.70% and 59.22%,respectively. The results of scanning electron microscopy showed that the mycelial growth of R. solani treated with GXLL3319 was obviously inhibited,and the mycelia were fractured,swollen and dissolving. Thirty-one antibacterial active substances was identified from VOCs,including ketones,alcohols,aldehydes,heterocycles,sulfides, organic acids,esters,alkanes and phenols. The results of pot experiment showed that GXLL3319 bacterial fluid could effectively inhibit the occurrence of tobacco target spot,and the relative control efficacy reached 66.07%,which was not significantly different(P>0.05)from that of 15% Jinggangmycin A soluble powder.【Conclusion】B. subtilis GXLL3319 can effectively inhibit the mycelial growth of R. solani,its VOCs contain a variety of antibacterial active substances. GXLL3319 has a good control efficacy on tobacco target spot and has the potential to be developed as a biocontrol bacteria against tobacco target spot.
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